PROJECT SUMMARY This proposal is in response to the Funding Opportunity Announcement (PAR-16-369) ?Resource-Related Research Projects for Development of Animal Models and Related Materials (R24).? Our proposal addresses the goals stated in the Research Objectives: ?The grant supports research projects that contribute to the knowledge of a model system, making the system more useful and accessible to the research community.? Specifically, we will generate a resource of Drosophila cell lines, markers, and knockouts that will enable cell biological analysis of genes and pathways altered in human diseases, including in disease-relevant tissue lineages and genetic backgrounds. In recent years, we have witnessed renewed interest in Drosophila cell lines and it is now common for Drosophila researchers to go back and forth between in vivo and cell culture studies. However, the full potential of Drosophila cell lines, particularly for disease-related studies, has not yet been realized, as the diversity of cell lines and markers available is limited. To overcome this bottleneck, we propose to use exciting new advances in cell line immortalization and genome engineering to generate reagents that will enable the community to fully exploit the power of Drosophila cell lines to address disease-related cell biological questions. Specifically, we propose to: Aim 1: Generate new cell lines from specific Drosophila cell lineages that will enable screens and other studies in relevant tissue-derived cell lines; Aim 2: Use CRISPR genome engineering to generate a resource of cell lines expressing fluorescent markers of sub-cellular components that will enable the community to perform screens exploring organelles and cell biological events relevant to human disease; and Aim 3: Use CRISPR engineering to generate knockout cell lines for synthetic lethal screens and other applications. We will disseminate information and resources for creating cell disease models by making available to the community detailed protocols and plasmid vectors, as well as the new cell lines and derivatives. Importantly, as Drosophila is an established system for the study of diverse human diseases, the resource and tools that we will develop are relevant to nearly all NIH institutes. Finally, we document that these resources will have benefit beyond the Drosophila community as they can be used in a number of creative ways to uncover exciting new information about conserved gene function, protein complexes and interactions, organelle function under normal or perturbed conditions, and more.